Human placental organoids as a model to probe early gestation maternal immune dynamics.

During pregnancy, the human placenta establishes tolerance toward fetal allogeneic tissue, where specialized trophoblast subtypes play a complex role in local and peripheral immunomodulation. However, due to inadequate models to study the early gestation of the human placenta, each trophoblast subtype's role in modulating the maternal immune response has remained elusive. Here, we derived human placental organoids from early gestation trophoblast stem cells to (1) identify patterns of immunomodulatory protein expression by trophoblast subtype and (2) evaluate the effects of the placental organoid secretome on immune cell activation and regulation. We show that the three primary trophoblast phenotypes had distinct influences on immune cell phenotype and activation and that three-dimensional culture significantly alters trophoblast immunomodulation relative to traditional two-dimensional trophoblast culture.

Engineered 3D Hydrogel Matrices to Modulate Trophoblast Stem Cell-Derived Placental Organoid Phenotype.

Placental organoid models are a promising platform to study human placental development and function. Organoid systems typically use naturally derived hydrogel extracellular matrices (ECM), resulting in batch-to-batch variability that limits experimental reproducibility. As an alternative, synthetic ECM-mimicking hydrogel matrices offer greater consistency and control over environmental cues. Here, we generated trophoblast stem cell-derived placental organoids using poly(ethylene glycol) (PEG) hydrogels with tunable degradability and placenta-derived ECM cues to evaluate trophoblast differentiation relative to Matrigel and two-dimensional (2D) culture controls. Our data demonstrate that PEG hydrogels support trophoblast viability and metabolic function comparable to gold standard Matrigel. Additionally, phenotypic characterization via proteomic analysis revealed that PEG and Matrigel matrices drive syncytiotrophoblast and extravillous trophoblast-dominant placental organoid phenotypes, respectively. Further, three-dimensional (3D) environments promoted greater integrin expression and ECM production than 2D culture. This study demonstrates that engineered 3D culture environments can be used to reliably generate placental organoids and guide trophoblast differentiation.

pO2 reporter composite hydrogel macroencapsulation devices for magnetic resonance imaging oxygen quantification.

Hydrogel cell encapsulation devices are a common approach to reduce the need for chronic systemic immunosuppression in allogeneic cell product transplantation. Macroencapsulation approaches are an appealing strategy, as they maximize graft retrievability and cell dosage within a single device; however, macroencapsulation devices face oxygen transport challenges as geometries increase from preclinical to clinical scales. Device design guided by computational approaches can facilitate graft oxygen availability to encapsulated cells in vivo but is limited without accurate measurement of oxygen levels within the transplant site and graft. In this study, we engineer pO2 reporter composite hydrogels (PORCH) to enable spatiotemporal measurement of oxygen tension within macroencapsulation devices using the proton Imaging of siloxanes to map tissue oxygenation levels (PISTOL) magnetic resonance imaging approach. We engineer two methods of incorporating siloxane oximetry reporters within hydrogel devices, an emulsion and microbead-based approach, and evaluate PORCH cytotoxicity on co-encapsulated cells and accuracy in quantifying oxygen tension in vitro. We find that both emulsion and microbead PORCH approaches enable accurate in situ oxygen quantification using PISTOL magnetic resonance oximetry, and that the emulsion-based PORCH approach results in higher spatial resolution.

Controlling placental spheroid growth and phenotype using engineered synthetic hydrogel matrices.

The human placenta is a complex organ comprised of multiple trophoblast subtypes, and inadequate models to study the human placenta in vitro limit the current understanding of human placental behavior and development. Common in vitro placental models rely on two-dimensional culture of cell lines and primary cells, which do not replicate the native tissue microenvironment, or poorly defined three-dimensional hydrogel matrices such as Matrigel™ that provide limited environmental control and suffer from high batch-to-batch variability. Here, we employ a highly defined, synthetic poly(ethylene glycol)-based hydrogel system with tunable degradability and presentation of extracellular matrix-derived adhesive ligands native to the placenta microenvironment to generate placental spheroids. We evaluate the capacity of a hydrogel library to support the viability, function, and phenotypic protein expression of three human trophoblast cell lines modeling varied trophoblast phenotypes and find that degradable synthetic hydrogels support the greatest degree of placental spheroid viability, proliferation, and function relative to standard Matrigel controls. Finally, we show that trophoblast culture conditions modulate cell functional phenotype as measured by proteomics analysis and functional secretion assays. Engineering precise control of placental spheroid development in vitro may provide an important new tool for the study of early placental behavior and development.

Translation of cell therapies to treat autoimmune disorders.

Autoimmune diseases are a diverse and complex set of chronic disorders with a substantial impact on patient quality of life and a significant global healthcare burden. Current approaches to autoimmune disease treatment comprise broadly acting immunosuppressive drugs that lack disease specificity, possess limited efficacy, and confer undesirable side effects. Additionally, there are limited treatments available to restore organs and tissues damaged during the course of autoimmune disease progression. Cell therapies are an emergent area of therapeutics with the potential to address both autoimmune disease immune dysfunction as well as autoimmune disease-damaged tissue and organ systems. In this review, we discuss the pathogenesis of common autoimmune disorders and the state-of-the-art in cell therapy approaches to (1) regenerate or replace autoimmune disease-damaged tissue and (2) eliminate pathological immune responses in autoimmunity. Finally, we discuss critical considerations for the translation of cell products to the clinic.

Engineering of Trophoblast Extracellular Vesicle-Delivering Hydrogels for Localized Tolerance Induction in Cell Transplantation.

Purpose: The need for chronic systemic immunosuppression, which presents a host of acute risks to transplantation patients, remains the primary limitation for the translation of many cell therapies, such as insulin secreting cells for the treatment of type 1 diabetes. Trophoblasts are the professional tolerogenic cells of the placenta, and they secrete a range of soluble factors to induce antigen specific tolerance toward allogeneic fetal tissue during pregnancy, including extracellular vesicles. Here we develop a trophoblast extracellular vesicle-delivering hydrogel designed for sustained, localized tolerogenic factor delivery within a transplant site to induce localized tolerance toward cell grafts. Methods: We engineer a synthetic poly(ethylene glycol)-based hydrogel system to tether extracellular vesicles for sustained delivery, and compare this system to passive vesicle entrapment within an alginate hydrogel system. We characterize trophoblast extracellular vesicles for size and morphology, and evaluate vesicle tolerogenic protein content via proteomic analysis. We validate the retention and tethering of extracellular vesicles within the hydrogel systems via scanning electron and stimulated emission depletion microscopy, and measure vesicle release rate over time. Finally, we evaluate trophoblast extracellular vesicle influence on natural killer cell activation in vitro. Results: We isolated trophoblast extracellular vesicles and proteomics confirmed the presence of tolerogenic factors. We confirmed the presence of extracellular vesicles within hydrogel delivery vehicles, and synthetic hydrogels extended extracellular vesicle release relative to a passive hydrogel system. Finally, extracellular vesicles reduced natural killer cell activation in vitro, confirming the tolerogenic potential of hydrogel-delivered extracellular vesicles. Conclusions: This tolerogenic extracellular vesicle-delivering hydrogel platform designed for delivery within a transplant site could serve as an alternative to systemic immunosuppression in cell transplantation, potentially reducing the risks associated with cell therapies and widening the eligible patient population.

Engineering synthetic poly(ethylene) glycol-based hydrogels compatible with injection molding biofabrication.

Hydrogel injection molding is a biofabrication method that is useful for the rapid generation of complex cell-laden hydrogel geometries, with potential utility in biomanufacturing products for tissue engineering applications. Hydrogel injection molding requires that hydrogel polymers have sufficiently delayed crosslinking times to enable injection and molding prior to gelation. In this work, we explore the feasibility of injection molding synthetic poly(ethylene) glycol (PEG)-based hydrogels functionalized with strain promoted azide-alkyne cycloaddition click chemistry functional groups. We evaluate the mechanical properties of a PEG-based hydrogel library, including time to gelation and successful generation of complex geometries via injection molding. We evaluate the binding and retention of adhesive ligand RGD within the library matrices and characterize the viability and function of encapsulated cells. This work demonstrates the feasibility of injection molding synthetic PEG-based hydrogels for tissue engineering applications, with potential utility in the clinic and biomanufacturing.

Differential expression of CCR8 in tumors versus normal tissue allows specific depletion of tumor-infiltrating T regulatory cells by GS-1811, a novel Fc-optimized anti-CCR8 antibody.

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.

Hydrogel Injection Molding to Generate Complex Cell Encapsulation Geometries.

Biofabrication methods capable of generating complex, three-dimensional, cell-laden hydrogel geometries are often challenging technologies to implement in the clinic and scaled manufacturing processes. Hydrogel injection molding capitalizes on the reproducibility, efficiency, and scalability of the injection molding process, and we adapt this technique to biofabrication using a library of natural and synthetic hydrogels with varied crosslinking chemistries and kinetics. We use computational modeling to evaluate hydrogel library fluid dynamics within the injection molds in order to predict molding feasibility and cytocompatibility. We evaluate the reproducibility of hydrogel construct molding and extraction and establish criteria for the selection of hydrogels suitable for injection molding. We demonstrate that hydrogel injection molding is capable of generating complex three-dimensional cell-laden construct geometries using diverse hydrogel materials and that this platform is compatible with primary human islet encapsulation. These results highlight the versatility and feasibility of hydrogel injection molding as a biofabrication technique with potential applications in the clinic and biomanufacturing.

A Method for Organoid Transplantation and Whole-Mount Visualization of Post-Engraftment Vascularization.

As the field of organoid development matures, the need to transplant organoids to evaluate and characterize their functionality grows. Decades of research developing islet organoid transplantation for the treatment of type 1 diabetes can contribute substantially to accelerating diverse tissue organoid transplantation. Biomaterials-based organoid delivery methods offer the potential to maximize organoid survival and engraftment. In this protocol, we describe a vasculogenic degradable hydrogel vehicle and a method to deliver organoids to intraperitoneal tissue. Further, we describe a method to fluorescently label and image functional vasculature within the graft as a tool to investigate organoid engraftment.

Biomaterial-based approaches to engineering immune tolerance.

The development of biomaterial-based therapeutics to induce immune tolerance holds great promise for the treatment of autoimmune diseases, allergy, and graft rejection in transplantation. Historical approaches to treat these immunological challenges have primarily relied on systemic delivery of broadly-acting immunosuppressive agents that confer undesirable, off-target effects. The evolution and expansion of biomaterial platforms has proven to be a powerful tool in engineering immunotherapeutics and enabled a great diversity of novel and targeted approaches in engineering immune tolerance, with the potential to eliminate side effects associated with systemic, non-specific immunosuppressive approaches. In this review, we summarize the technological advances within three broad biomaterials-based strategies to engineering immune tolerance: nonspecific tolerogenic agent delivery, antigen-specific tolerogenic therapy, and the emergent area of tolerogenic cell therapy.

Publisher Correction: Phosphorylation of PD-1-Y248 is a marker of PD-1-mediated inhibitory function in human T cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunotherapy via PD-L1-presenting biomaterials leads to long-term islet graft survival.

Antibody-mediated immune checkpoint blockade is a transformative immunotherapy for cancer. These same mechanisms can be repurposed for the control of destructive alloreactive immune responses in the transplantation setting. Here, we implement a synthetic biomaterial platform for the local delivery of a chimeric streptavidin/programmed cell death-1 (SA-PD-L1) protein to direct "reprogramming" of local immune responses to transplanted pancreatic islets. Controlled presentation of SA-PD-L1 on the surface of poly(ethylene glycol) microgels improves local retention of the immunomodulatory agent over 3 weeks in vivo. Furthermore, local induction of allograft acceptance is achieved in a murine model of diabetes only when receiving the SA-PD-L1-presenting biomaterial in combination with a brief rapamycin treatment. Immune characterization revealed an increase in T regulatory and anergic cells after SA-PD-L1-microgel delivery, which was distinct from naïve and biomaterial alone microenvironments. Engineering the local microenvironment via biomaterial delivery of checkpoint proteins has the potential to advance cell-based therapies, avoiding the need for systemic chronic immunosuppression.

Targeted deletion of PD-1 in myeloid cells induces antitumor immunity.

PD-1, a T cell checkpoint receptor and target of cancer immunotherapy, is also expressed on myeloid cells. The role of myeloid-specific versus T cell-specific PD-1 ablation on antitumor immunity has remained unclear because most studies have used either PD-1-blocking antibodies or complete PD-1 KO mice. We generated a conditional allele, which allowed myeloid-specific (PD-1f/fLysMcre) or T cell-specific (PD-1f/fCD4cre) targeting of Pdcd1 gene. Compared with T cell-specific PD-1 ablation, myeloid cell-specific PD-1 ablation more effectively decreased tumor growth. We found that granulocyte/macrophage progenitors (GMPs), which accumulate during cancer-driven emergency myelopoiesis and give rise to myeloid-derived suppressor cells (MDSCs), express PD-1. In tumor-bearing PD-1f/fLysMcre but not PD-1f/fCD4cre mice, accumulation of GMP and MDSC was prevented, whereas systemic output of effector myeloid cells was increased. Myeloid cell-specific PD-1 ablation induced an increase of T effector memory cells with improved functionality and mediated antitumor protection despite preserved PD-1 expression in T cells. In PD-1-deficient myeloid progenitors, growth factors driving emergency myelopoiesis induced increased metabolic intermediates of glycolysis, pentose phosphate pathway, and TCA cycle but, most prominently, elevated cholesterol. Because cholesterol is required for differentiation of inflammatory macrophages and DC and promotes antigen-presenting function, our findings indicate that metabolic reprogramming of emergency myelopoiesis and differentiation of effector myeloid cells might be a key mechanism of antitumor immunity mediated by PD-1 blockade.

Publisher Correction: Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phosphorylation of PD-1-Y248 is a marker of PD-1-mediated inhibitory function in human T cells.

PD-1 is a target of cancer immunotherapy but responses are limited to a fraction of patients. Identifying patients with T cells subjected to PD-1-mediated inhibition will allow selection of suitable candidates for PD-1-blocking therapy and will improve the therapeutic success. We sought to develop an approach to detect PD-1-mediated inhibitory signaling. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) encompassing Y223 and an immunoreceptor tyrosine-based switch motif (ITSM) encompassing Y248, which is indispensable for interaction of SHP-2 and delivery of PD-1 inhibitory function. We generated an antibody specific for phosphorylated PD-1-Y248 and examined PD-1pY248+ (pPD-1) expression in human T cells. pPD-1 was upregulated by TCR/CD3 + CD28 stimulation and simultaneous PD-1 ligation. pPD-1+CD8+ T cells were identified in human peripheral blood and had impaired effector function. pPD-1+ T cells were also detected in tumor-draining lymph nodes of tumor bearing mice and in biopsies of patients with glioblastoma multiform. Detection of pPD-1+ T cells might serve as a biomarker for identification of T cells subjected to PD-1-mediated immunosuppression.

Linkage Groups within Thiol-Ene Photoclickable PEG Hydrogels Control In Vivo Stability.

Thiol-norbornene (thiol-ene) photoclickable poly(ethylene glycol) (PEG) hydrogels are a versatile biomaterial for cell encapsulation, drug delivery, and regenerative medicine. Numerous in vitro studies with these 4-arm ester-linked PEG-norbornene (PEG-4eNB) hydrogels demonstrate robust cytocompatibility and ability to retain long-term integrity with nondegradable crosslinkers. However, when transplanted in vivo into the subcutaneous or intraperitoneal space, these PEG-4eNB hydrogels with nondegradable crosslinkers rapidly degrade within 24 h. This characteristic limits the usefulness of PEG-4eNB hydrogels in biomedical applications. Replacing the ester linkage with an amide linkage (PEG-4aNB) mitigates this rapid in vivo degradation, and the PEG-4aNB hydrogels maintain long-term in vivo stability for months. Furthermore, when compared to PEG-4eNB, the PEG-4aNB hydrogels demonstrate equivalent mechanical properties, crosslinking kinetics, and high cytocompatibility with rat islets and human mesenchymal stem cells. Thus, the PEG-4aNB hydrogels may be a suitable replacement platform without necessitating critical design changes or sacrificing key properties relevant to the well-established PEG-4eNB hydrogels.

The regulation of ZCT1, a transcriptional repressor of monoterpenoid indole alkaloid biosynthetic genes in Catharanthus roseus.

Cys2/His2-type (C2H2) zinc finger proteins, such as ZCT1, are an important class of transcription factors involved in growth, development, and stress responses in plants. In the medicinal plant Catharanthus roseus, the zinc finger Catharanthus transcription factor (ZCT) family represses monoterpenoid indole alkaloid (MIA) biosynthetic gene expression. Here, we report the analysis of the ZCT1 promoter, which contains several hormone-responsive elements. ZCT1 is responsive to not only jasmonate, as was previously known, but is also induced by the synthetic auxin, 1-naphthalene acetic acid (1-NAA). Through promoter deletion analysis, we show that an activation sequence-1-like (as-1-like)-motif and other motifs contribute significantly to ZCT1 expression in seedlings. We also show that the activator ORCA3 does not transactivate the expression of ZCT1 in seedlings, but ZCT1 represses its own promoter, suggesting a feedback mechanism by which the expression of ZCT1 can be limited.

Synthetic poly(ethylene glycol)-based microfluidic islet encapsulation reduces graft volume for delivery to highly vascularized and retrievable transplant site.

Transplant of hydrogel-encapsulated allogeneic islets has been explored to reduce or eliminate the need for chronic systemic immunosuppression by creating a physical barrier that prevents direct antigen presentation. Although successful in rodents, translation of alginate microencapsulation to large animals and humans has been hindered by large capsule sizes (≥500 µm diameter) that result in suboptimal nutrient diffusion in the intraperitoneal space. We developed a microfluidic encapsulation system that generates synthetic poly(ethylene glycol)-based microgels with smaller diameters (310 ± 14 µm) that improve encapsulated islet insulin responsiveness over alginate capsules and allow transplant within vascularized tissue spaces, thereby reducing islet mass requirements and graft volumes. By delivering poly(ethylene glycol)-encapsulated islets to an isolated, retrievable, and highly vascularized site via a vasculogenic delivery vehicle, we demonstrate that a single pancreatic donor syngeneic islet mass exhibits improved long-term function over conventional alginate capsules and close integration with transplant site vasculature. In vivo tracking of bioluminescent allogeneic encapsulated islets in an autoimmune type 1 diabetes murine model showed enhanced cell survival over unencapsulated islets in the absence of chronic systemic immunosuppression. This method demonstrates a translatable alternative to intraperitoneal encapsulated islet transplant.

Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

ß-cell proliferation induction is a promising therapeutic strategy to restore ß-cell mass. By screening small molecules in a transgenic zebrafish model of type 1 diabetes, we identified inhibitors of non-canonical IκB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε), as enhancers of ß-cell regeneration. The most potent ß-cell regeneration enhancer was a cinnamic acid derivative (E)-3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA), which, acting through the cAMP-dependent protein kinase A (PKA), stimulated ß-cell-specific proliferation by increasing cyclic AMP (cAMP) levels and mechanistic target of rapamycin (mTOR) activity. A combination of PIAA and cilostamide, an inhibitor of ß-cell-enriched cAMP hydrolyzing enzyme phosphodiesterase (PDE) 3, enhanced ß-cell proliferation, whereas overexpression of PDE3 blunted the mitogenic effect of PIAA in zebrafish. PIAA augmented proliferation of INS-1ß-cells and ß-cells in mammalian islets including human islets with elevation in cAMP levels and insulin secretion. PIAA improved glycemic control in streptozotocin (STZ)-induced diabetic mice with increases in ß-cell proliferation, ß-cell area, and insulin content in the pancreas. Collectively, these data reveal an evolutionarily conserved and critical role of TBK1/IKKε suppression in expanding functional ß-cell mass.